Specificity and stability of the chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L) Dc).
The specificity of the winged bean chymotrypsin inhibitor is restricted to the chymotrypsins (EC 3.4.21.1 and EC 3.4.21.2). Trypsins (EC 3.4.21.4), elastase (EC 3.4.21.11), subtilisins (EC 3.4.21.14), proteinase K (EC 3.4.21.14) and Pronase (EC 3.4.24.4) are not inhibited. The inhibitor reacts with two molecules of chymotrypsin to form a stable complex (Mr approx. 70 0000) which was isolated by gel filtration on Sephadex G-100. When mixed with substrate, the interaction of the inhibitor with alpha-chymotrypsin is characterized by substrate-induced dissociation of the complex. In contrast, the interaction with chymotrypsin B is quantitative with no substrate-induced dissociation. The inhibitor reacts with alpha-chymotrypsin to form a 1 : 2 molar complex at all ratios of [I]/[E]; however, the interaction with chymotrypsin B is characterized by the formation of initially of a 1 : 1 molar complex at [I] greater than [E] followed by the formation of the 1 : 2 molar complex at [I] less than 2[E]; an intermediate species of Mr approx. 48 000 was demonstrated by gel filtration on Sephadex G-100. The inhibitor is stable over the pH range 2.0-11.5 and to heating up to 70 degrees C at pH 4.1 and 8.0, and up to 90 degrees C at pH 3.0. The inhibitor resists denaturation in 8.0 M urea at pH 8.0 and 4.0, is stable in 0.12 M beta-mercaptoethanol at pH 8.0; however, reduction in 8.0 M urea results in a loss of inhibitory activity. The inhibitor resists digestion with pepsin at pH 2.0, being only slowly degraded over a period of 7 days with an equimolar amount of pepsin.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010434:Pepsin A / Q000502:physiology; D010946:Plants, Medicinal; D012639:Seeds / Q000032:analysis; D013379:Substrate Specificity; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D008623:Mercaptoethanol; D014508:Urea; D002918:Chymotrypsin; D010434:Pepsin A